Bioluminescence refers to the emission of light by biological molecules. Bioluminescent proteins can be true enzymes such as luciferases, which catalyze the oxidation of luciferin, emitting light and releasing oxyluciferin, or photoproteins, which catalyze the oxidation of luciferin to emit light but do not release the oxidized substrate. Examples of bioluminescent proteins include those isolated from the ctenophores Mnemiopsis (mnemiopsin) and Beroe ovata (berovin), those isolated from the coelenterates Aequoria (aequorin), Obelia (obelin), Pelagia, and luciferases such as Renilla (Renilla luciferase) and those isolated from the molusca Pholas (pholasin). Bioluminescent proteins also can be isolated from ostracods such as Cypridina.
A bioluminescent protein particularly useful as a label in diagnostic assays is the photoprotein aequorin. Aequorin catalyzes the oxidation of coelenterate luciferin to oxyluciferin, resulting in the concomitant production of blue light (lambda.sub.max =469 nm). Aequorin consists of apoaequorin, a single polypeptide chain of M.sub.r 22,000, containing one mole each of tightly bound coelenterate luciferin and oxygen. Light emission from this complex is initiated upon the binding of calcium ions. Aequorin catalyzes a single turnover event or "flash reaction" which persists for approximately 10 seconds.
Due to the relatively high quantum yield of the reaction, aequorin can be detected at the attomol level (10.sup.-18 mol) using commercially available luminometers. Because of its calcium requirement and ability to be detected at very low levels, aequorin has proven useful for monitoring levels of intracellular calcium. The potential utility of using aequorin as a nonradioisotopic reporter molecule has only recently been realized with the availability of the recombinant protein (reviewed by Cormier et al., Cormier, M. J. Prasher DC Longiatu, M & McCann, R. O. 1989, Photochem. Photobiol. 49:509-512).
Until very recently, it was generally considered in the art that all bioluminescent proteins were inherently unstable and complex, thus making them unsuitable for reporter molecules, based on the properties of firefly Photinus pyralis luciferase (Bronstein & McGrath, 1989 Chemiluminescence Lights Up. Nature 338, 599-600). However, the biotinylated derivative of aequorin recently has been shown to retain greater than 80 percent of its original activity and, when used in combination with streptavidin, has been demonstrated to detect nanogram to subnanogram amounts of biotinylated targets, including protein antigens and DNA immobilized onto microtiter walls or membrane supports (Stults et al., 1992, Rivera, H., McCann, R. O., O'Kane D., Cummings, R. D., Cormier M. J., Smith D. F. Use of Recombinant Biotinylated-Aequorin in Microtiter and Membrane-Based Assays. Purification of Recombinant Apoaequorin From Escherichia coli. Biochemistry, 31, 1433-1442). In addition, biotinylated aequorin also has been successfully used in a capture immunoassay for Salmonella antigen (Smith et al., 1991, A Microplate Assay For Analysis of Solution Phase Glycosyltransferase Reactions: Determination of Kinetic Constants. Anal. Biochem. 199, 286-292) and in solid phase assays for glycosyl transferases and glycoproteins (Mengeling et al., 1991, A Microplate Assay For Analysis of Solution Phase Glycosyltransferase Reactions: Determination of Kinetic Constants. Anal. Biochem. 199, 286-292 ), (Zatta et al., 1991, A Solid Phase Assay For .beta.1,4 Galactosyl Transferase Activity in Human Serum Using Recombinant Aequorin, Anal. Biochem. 194, 185-191).
Although the biotinylated derivative of aequorin performs well in a variety of different assay formats, it would be advantageous to synthesize by chemical crosslinking methods direct conjugates of recombinant aequorin with a variety of selectively bindable reagent including receptors, hormones, lectins, antibodies and binding fragments thereof, antigens, DNA, RNA, oligonucleotides, and glycoproteins. Such conjugates would facilitate faster detection of biological targets by reducing the number of incubation steps or by eliminating the avidin/streptavidin-biotin interaction entirely.
Direct chemical crosslinking of apoaequorin to antibody FAB fragments using a heterobifunctional reagent has been reported (Erikaku et al., 1991, Bioluminescent Immunoassay Using a Monomeric Fab'-Photoprotein Aequorin Conjugate. Biochem. Biophys. Res. Commun. 174, 1331-1336). The antibody conjugate was prepared using a chemical crosslinking technique based on modification of amino or thiol groups on apoaequorin with maleimido groups. Unfortunately, this crosslinking procedure results in apoaequorin-antibody conjugates that retained about only 10 percent of the activity of underivatized aequorin. (See Abst., p. 1331 Erikaku et al, 1991).
What is needed, therefore, is a direct chemical crosslinking method for preparing photoprotein conjugates that substantially preserves photoprotein activity.